Growth factor-binding compounds and methods of use

ABSTRACT

Growth factor binding compounds having a plurality of acyclic isophthalic acid groups attached to a non-peptide organic scaffold and pharmaceutical compositions of the same are disclosed. Methods of administering and using the growth factor binding compounds or the growth factor binding compositions are also taught. These novel growth factor binding compounds are useful for treating angiogenesis, excessive cellular proliferation, tumor growth, and a combination thereof as well as inhibiting growth factor binding to cells and phosphorylation.

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims benefit of U.S. Provisional ApplicationSer. No. 60/539,613, filed Jan. 27, 2004, which is hereby incorporatedby reference herein in its entirety, including any figures, tables,nucleic acid sequences, amino acid sequences, and drawings.

The subject invention was made with government support under a researchproject supported by National Institute of Health/National CancerInstitute Grant No. CA78038. The federal government may have certainrights in this invention.

BACKGROUND OF THE INVENTION

The ability of tumors to grow beyond a few cubic millimeters in volumedepends on the formation of new blood vessels within themicroenvironment of the tumors (Ferrara, N. Nat Rev Cancer, 2002,2:795-803; Kerbel, R. S. Carcinogenesis, 2000, 21:505-15; Carmeliet, P.and Jain, R. K. Nature, 2000, 407:249-57; Yancopoulos, G. D. et al.Nature, 2000, 407:242-8). This angiogenic process is triggered byseveral key growth factors that are secreted by the tumor. The growthfactors not only bind their receptors on endothelial cells and stimulatetheir proliferation initiating new blood vessel formation, but also bindreceptors on accessory cells such as pericytes that maintain vesselintegrity (Ferrara, N. Nat Rev Cancer, 2002, 2:795-803; Kerbel, R. S.Carcinogenesis, 2000, 21:505-15; Carmeliet, P. and Jain, R. K. Nature,2000, 407:249-57; Yancopoulos, G. D. et al. Nature, 2000, 407:242-8;Helmlinger, G., et al. Nat Med, 1997, 3:177-82; Holash, J. et al.Science, 1999, 284:1994-8). Among the most studied growth factors arevascular endothelial growth factor (VEGF) and platelet-derived growthfactor (PDGF). Several studies have demonstrated the participation ofthese two growth factors in the angiogenic process with VEGF playing akey role mainly in the initiation of the formation of new blood vesselsand PDGF being involved in the maintenance of these vessels (Bergers, G.et al. J Clin Invest, 2003, 111:1287-95; Dvorak, H. F. J Clin Oncol,2002, 20:4368-80; Ferrara, N. Curr Top Microbiol Immunol, 1999,237:1-30; Dvorak, H. F. et al. Curr Top Microbiol Immunol, 1999,237:97-132; Eriksson, U. and Alitalo, K. Curr Top Microbiol Immunol,1999, 237:41-57).

This observation prompted an interest in designing strategies tosuppress the functions of VEGF and PDGF, with the ultimate goal ofinhibiting angiogenesis and starving tumors. The approaches that havebeen taken were based on targeting the biochemical steps involved in themechanism of action of these growth factors. These include inhibitingthe binding of VEGF and PDGF to their respective receptors by usingantibodies against the growth factors. One of these, AVASTIN, whichtargets VEGF, has recently been approved for clinical use in patientswith metastatic colorectal cancer (Zhang, W. et al. Angiogenesis, 2002,5:35-44; Ferrara, N. Semin Oncol, 2002, 29:10-4). Another approach hasinvolved the development of inhibitors of the tyrosine kinase activitiesof the PDGF and VEGF receptors, resulting in suppression of thedownstream signal transduction pathways triggered by these growthfactors (Kerbel, R. S. Carcinogenesis, 2000, 21:505-15; Jain, R. K.Semin Oncol, 2002, 29:3-9; Morin, M. J. Oncogene, 2000, 19:6574-83;Miao, R. Q. et al. Blood, 2002, 100:3245-52; Laird, A. D. et al. CancerRes, 2000, 60:4152-60; Wedge, S. R. et al. Cancer Res, 2000, 60:970-5).Most of these agents mimic the structure of ATP and some are potentantitumor agents that are presently in clinical trials. However, nonehave been approved yet by the FDA.

The approval by the FDA of AVASTIN (bevacizumab), which increases by 5months the median survival of patients with metastatic colorectalcancer, further validates targeting angiogenic processes as a strategyto treat cancer (Ferrara, N. Semin Oncol, 2002, 29:10-4). However, muchmore needs to be done to fully exploit this approach. For example, inother clinical trials, AVASTIN failed to prolong the lives of patientswith metastatic breast cancer. One possible explanation for thisinconsistent activity is that advanced metastatic breast cancer maycircumvent anti-VEGF angiogenesis therapy by means of other growthfactors. Indeed support for this suggestion comes from preclinicalstudies showing that early breast cancer secretes mainly VEGF whereasadvanced breast cancer secretes additional growth factors (Relf, M. etal. Cancer Res, 1997, 57:963-9). Furthermore, in an animal pancreaticcancer model, SU5416, a VEGF receptor tyrosine kinase inhibitorsuppresses early, but not late, development of pancreatic tumors. Moreimportantly in the same model, treatment with SU6668 (which inhibitsboth VEGF and PDGF receptor tyrosine kinases) induced regression ofadvanced pancreatic tumor at late stage of development (Bergers, G. etal. J Clin Invest, 2003, 111:1287-95) suggesting that the failure ofanti-VEGF therapy may be due to its ability to inhibit only initiationbut not maintenance of blood vessels. Further support for thissuggestion comes from a very recent study where AVASTIN inhibited theformation of new blood vessels but was ineffective at inhibiting alreadyestablished ones in an animal model where neuroblastoma cells weretransplanted onto mouse kidneys (Huang, J. et al. Proc Natl Acad SciUSA, 2003, 100:7785-90). Taken together, the present understanding ofthe angiogenesis process suggests that simultaneously targeting ofgrowth factors that initiate (i.e., VEGF) as well as those that maintain(i.e., PDGF) blood vessels may be a more effective approach to cancertherapy than targeting only one growth factor.

BRIEF SUMMARY OF THE INVENTION

It is an object of the subject invention to design a family of compoundsthat bind VEGF and/or PDGF and inhibit the binding of these growthfactors to their respective cell surface receptors. For example, thecompound GFB204, was found to be a potent and selective inhibitor ofVEGF- and PDGF-stimulation of their receptor tyrosine kinasephosphorylation and signaling (Erk1/2, Akt and STAT3). Thispharmacological agent also potently inhibited endothelial cell migrationand capillary network formation in vitro as well as in vivo blood vesselformation and human tumor growth in nude mouse xenografts.

It is a further object of the subject invention to providepharmaceutical compositions of the above-referenced family of compoundsand methods of administering the same.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows structures of GFB204 of the present invention, which haveacyclic isophthalic acid groups attached to a non-peptide organicscaffold as well as GFB-111.

FIGS. 2A-2C show that GFB204 inhibits ¹²⁵I-VEGF and ¹²⁵I-PDGF but not¹²⁵I-EGF binding to their receptors in mouse fibroblasts. Flk-1/NIH 3T3,NIH 3T3 and EGFR/NIH 3T3 cells were incubated with ¹²⁵I-VEGF, ¹²⁵I-PDGFand ¹²⁵I-EGF (50,000 cpm/well) respectively, along with increasingconcentrations of GFB204. Cells were incubated at 4° C. for 0.5 hours,then washed three times with PBS and three times with lysis buffer priorto determining ¹²⁵I counts as described under Materials and Methods. Anexcess of cold VEGF, PDGF, and EGF was used to obtain non-specificbinding levels. FIGS. 2A-2C show specific binding (% control) for PDGFR,Flk-1, and EGFR, respectively.

FIGS. 2D and 2E illustrate that GFB-204 binds PDGF and VEGF as indicatedby growth factor tryptophan when increased amounts of GFB204 were addedto PDGF and VEGF, respectively. The fluorescence was monitored byexcitation at 295 nm and 228 nm, respectively, and emission at 334 nmn.

FIGS. 3A and 3B show the effect of GFB204 on growth factor stimulatedErk1, Erk2, Akt, and STAT3 phosphorylation. GFB204 inhibits VEGF andPDGF stimulation of Flk-1 tyrosine phosphorylation and Erk1/Erk2phosphorylation (FIG. 3A). NIH 3T3 cells or Flk-1/NIH 3T3 cells weretreated with increasing concentrations of GFB204 for 5 minutes prior tostimulation with PDGFBB (10 ng/ml) or VEGF (50 ng/ml), respectively, for10 minutes. The cells were then lysed and processed for SDS-PAGE Westernblotting with an antibody specific for phosphotyrosine-Flk-1 oranti-phosphotyrosine for PDGFR tyrosine phosphorylation orphospho-Erk1/2. GFB204 effects on growth factor-stimulated Erk1, Erk2,Akt and STAT3 phosphorylation (FIG. 3B). NIH 3T3, Flk-1/NIH 3T3,IGF-R/NIH 3T3 or EGFR/NIH 3T3 cells were treated with GFB204 (10 μM)prior to stimulation with PDGF (NIH 3T3) VEGF (Flk-1/NIH 3T3), EGF(EGFR/NIH 3T3), bFGF (NIH 3T3) or IGF-1 (IGF-1R/NIH 3T3). The cells werethen harvested and processed for SDS-PAGE Western blotting withantibodies specific for phospho-Erk1/2, phospho-Akt and phospho-STAT3.

FIGS. 4A-4C show the effects of GFB204 on angiogenesis in vitro. GFB204inhibits capillary network formation in a dose-response manner (FIG.4C). Human middle cerebral artery endothelial cells (5×10⁴) were seededonto Matrigel and the cells were incubated with VEGF in the presence(FIG. 4B) or absence (FIG. 4A) of GFB204 as described under Materialsand Methods.

FIGS. 5A-5C illustrates that GFB204 potently inhibits VEGF-dependenthuman brain endothelial cell migration in vitro. Migration of adulthuman brain endothelial cells was evaluated using a modified Boydenchamber assay as described in Materials and Methods. Vehicle control(FIG. 5A) or GFB204 (FIG. 5B) was added to 2% FBS-containing medium inthe outer chamber, and the number of migrated cells to theVEGF-containing lower chamber was determined after an 18-hour incubation(FIG. 5C).

FIG. 6 illustrates that GFB204 inhibits A-549 xenografts growth in nudemice (FIG. 6). A-549 cells were implanted into the flanks of nude miceand when the tumors reached an average size of about 100 mm³, the micewere randomized and treated either with vehicle (♦) or GFB204 at 1 mg/kg(▪) and 5 mg/kg (●), and tumor sizes measured as described underMaterials and Methods. Tumors were processed two hours after the lasti.p. injection for CD31 IHC staining as described under Materials andMethods.

FIGS. 7A-7B illustrates CD31 IHC staining as described under Materialsand Methods for tumors processed two hours after the last i.p. injectionfor a control (FIG. 7A) and GFB204 (FIG. 7B). Quantification ofmicrovessels density (400×) was determined as described under Materialsand Methods. SE, standard error. FIGS. 7A and 7B Microvessel Count; FIG.7A=11.3±1.9; FIG. 7B=2.6±0.9.

MATERIALS AND METHODS

Inhibition of Growth Factor-Dependent Receptor Tyrosine Phosphorylationby GFBs.

Starved Flk-1/KDR-overexpressing NIH 3T3 cells (Flk-1/NIH 3T3) or NIH3T3 cells were pretreated with GFBs for 5 min before stimulation withVEGF (50 ng/ml) or PDGF-BB (10 ng/ml) for 10 min, respectively. Thecells were then harvested and lysed, and proteins from the lysates wereseparated by SDS-PAGE and transferred to nitrocellulose. Membranes thenwere either immunoblotted with anti-phospho-VEGFR2 antibody (CellSignaling Technologies, Beverly, Mass.) for activated Flk-1 oranti-phospho-tyrosine antibody (4G10, Upstate Biotechnology, LakePlacid, N.Y.) for activated PDGFR. Phosphotyrosine Flk-1 and PDGFR werequantified using a Bio-Rad Model GS-700 Imaging Densitometer (Bio-RadLaboratories, Inc, Hercules, Calif.) (Blaskovich, M. A. et al. NatBiotechnol, 2000, 18:1065-70).

Growth Factor-Mediated Stimulation of Phosphorylation of Erk1/2, Akt andSTAT3.

Starved NIH 3T3 cells (PDGF-BB, bFGF), NIH 3T3 cells overexpressing EGFR(EGFR/NIH 3T3, EGF), Flk-1 (Flk-1/NIH 3T3, VEGF), and IGF-1R NIH 3T3(IGF-1R/NIH 3T3, IGF-1) were pretreated with the indicated concentrationof GFB204 for 5 minutes before 10 minute stimulation with PDGF-BB (10ng/ml), EGF (10 ng/ml), bFGF (50 ng/ml), VEGF (50 ng/ml) and IGF-1 (50ng/ml). Cell lysates were run on SDS-PAGE gels, then transferred tonitrocellulose and Western blotted with anti-phosphorylated Erk1/Erk2(Cell Signaling Technologies) anti-phosphorylated Akt oranti-phosphorylated STAT3 as described previously by us (Blaskovich, M.A. et al. Cancer Res, 2003, 63:1270-9).

Binding of ¹²⁵I-growth factors to their receptors. The binding assay of¹²⁵I-VEGF, ¹²⁵I-PDGF and ¹²⁵I-EGF to their respective receptors wascarried out as described previously (Blaskovich, M. A. et al. NatBiotechnol, 2000, 18:1065-70. Briefly, Flk-1/NIH 3T3 cells, NIH 3T3cells and EGFR/NIH 3T3 cells were incubated with ¹²⁵I-VEGF, ¹²⁵I-PDGFand ¹²⁵I-EGF (50,000 cpm/well), respectively, and increasingconcentrations of GFB204. Cells were incubated at 4° C. for 0.5 hours,then washed three times with PBS and three times with 25 mM Tris, pH8.0, 1% Triton-X-100, 10% glycerol, and 1% SDS prior to determining ¹²⁵Icounts on a gamma counter (Beckmann Inc.). An excess of cold growthfactors were used to obtain nonspecific binding levels.

Capillary network formation. 200 Ξl of Matrigel was placed into eachwell of a 24-well culture plate at 4° C. and allowed to polymerize byincubation at 37° C. as described previously (Papadimitriou, E. et al.Biochem Biophys Res Commun, 2001, 282:306-13). Human middle cerebralartery endothelial cells (5×10⁴) were seeded on the Matrigel in 1 ml ofEBM containing VEGF (20 ng/ml). The cells were incubated in the presenceor absence of GFB204 at the concentrations indicated in the figurelegend. Each sample was photographed using a 10× objective lens, andquantified the total length of tube structures in each photograph usingthe Image Pro Plus software (Media Cybernetic, Inc., MD).

Human brain endothelial cell migration assay. Migration of adult humanbrain endothelial cells was evaluated using a modified Boyden chamberassay (BD BioCoat Matrigel Invasion Chamber) (Papadimitriou, E. et al.Biochem Biophys Res Commun, 2001, 282:306-13). The cells were plated at4×10⁴/ml onto an 8 μm pore size membrane coated with a thin layer ofMatrigel basement membrane matrix. GFB204 was added to the medium in theouter chamber and the cells were cultured for 18 hours underVEGF-dependent condition in the lower chamber (VEGF 20 ng/ml).Non-invading cells were removed from the upper surface with a cottonswab. Membrane inserts were then fixed with 4% paraformaldehyde andstained with Crystal-Violet dye. The number of cells that migrated tothe undersurface of the filters, was quantified by counting the cellsmigrated in randomly selected microscopic fields (10×). Samples wereanalyzed for significant differences using a Student's t-test forindependent samples.

Antitumor activity in the nude mouse tumor xenograft model. Nude mice(Charles River, Wilmington, Mass.) were maintained in accordance withthe Institutional Animal Care and Use Committee (IACUC) procedures andguidelines. A-549 cells were harvested and resuspended in PBS, theninjected s.c. into the right and left flanks (10×10⁶ cells per flank) of8 week old female nude mice as reported previously (Sun, J. et al.Cancer Res, 1999, 59:4919-26). When tumors reached about 100 mm³,animals were dosed i.p. with 0.2 ml solution once daily. Control animalsreceived a vehicle whereas treated animals were injected with GFB204 (1or 5 mg/kg/day). The tumor volumes were determined by measuring thelength (1) and the width (w) and calculating the volume (V=1 w²/2) asdescribed previously (Sun, J. et al. Cancer Res, 1999, 59:4919-26).Statistical significance between control and treated animals wereevaluated using Student's t-test.

IHC study. On the termination day of antitumor experiments, the tumorswere extracted and fixed in 10% neutral buffered formalin for 6 hours.After fixation, the tissue samples were processed into paraffin blocks.Tissue sections (4 μm thick) were obtained from the parablocks andstained with hematoxylin and eosin (H&E) using standard histologicaltechniques. Tissue sections were also subjected to immunostaining forCD31 (BD Biosciences, San Diego, Calif.) using the avidin biotinperoxidase complex technique (Blaskovich, M. A. et al. Nat Biotechnol,2000, 18:1065-70). Mouse monoclonal antibody was used at 1:50 dilution,following microwave antigen retrieval (four cycles of 5 minutes each onhigh in 0.1 M citrate buffer).

DETAILED DISCLOSURE OF THE INVENTION

The present invention pertains to growth factor-binding compounds. Moreparticularly, the present invention pertains to compounds (such as thoseshown in Table 1) that bind growth factors such as VEGF and/or PDGF, andare capable of inhibiting the binding of one or more of these growthfactors to their respective cell surface receptors. The invention alsoconcerns pharmaceutical compositions comprising one or more of thesecompounds and a pharmaceutically acceptable carrier.

In addition, the present invention concerns methods for inhibiting thebinding of such growth factors to cells by contacting one or morecompounds of the invention (or compositions comprising one or more ofthe compounds) with the cells in vitro or in vivo. In other aspects, thepresent invention includes methods for inhibiting growthfactor-stimulated phosphorylation (e.g., phosphorylation of Erk1, Erk2,Akt, and/or STAT3); methods for inhibiting angiogenesis; and methods forinhibiting cancer and/or tumor growth by contacting one or morecompounds or compositions of the present invention with target cells invitro or in vivo.

In a specific embodiment, the present invention concerns a method usefulfor inhibiting growth factors from binding to cells, for inhibitinggrowth factor stimulated phosphorylation, for inhibiting angiogenesis,for inhibiting cancer and tumor growth or a combination thereof, whereinthe method comprises contacting at least one growth factor bindingcompounds or a pharmaceutically acceptable salt of any of the growthfactor binding compounds, to a cell in vitro or in vivo; wherein thegrowth factor binding compounds comprise a plurality of acyclicisophthalic acid groups attached to a non-peptide organic scaffold;wherein each of the growth factor binding compounds, or thepharmaceutically salt of any of the growth factor binding compounds mayor may not be carried in a pharmaceutically acceptable carrier, exceptfor the compound having the general structure:

wherein each R1 is:

and each R2 is:

In a yet another specific embodiment, a pharmaceutical composition ofthe present invention is administered locally or systemically to apatient to achieve inhibition of angiogenesis, inhibition of tumorgrowth, and/or inhibition of cancer.

In one embodiment, the present invention includes a growthfactor-binding compound comprising a plurality of acyclic isophthalicacid groups attached to a non-peptide organic scaffold. In a furtherembodiment, the organic scaffold is a calix[4]arene scaffold. Acyclicisophthalic acid groups of the compounds of the invention can befunctionalized with an acidic group, a hydrophobic group, or both.

In another embodiment, the growth factor binding compound of the presentinvention has the general structure:

wherein each R₁ is independently selected from among the followingchemical groups:

and each R₂ is independently selected from among the following chemicalgroups:

In specific embodiments, the compound of the present invention isselected from the group consisting of GFB201, GFB202, GFB203, GFB204,GFB205, GFB206, GFB207, GFB208, GFB209, GFB210, GFB211, GFB212, GFB213,GFB214, GFB215, GFB216, GFB217, GFB218, and GFB219 (as set forth inTable 1).

Growth factors that are targeted or acted upon by the compounds of thesubject invention can include, but are not limited to, platelet derivedgrowth factor, a vascular endothelial growth factor, or both.

In another aspect, the present invention provides a compositioncomprising at least one compound of the invention, as disclosed herein,or a pharmaceutically acceptable salt thereof; and a pharmaceuticallyacceptable carrier.

In another aspect, the present invention provides a method of treating apatient having a disease comprising excess cellular proliferation,excess angiogenesis, a tumor, or a combination of any of the foregoing,wherein the method comprises administering to the patient an effectiveamount of a compound or composition of the invention. In a specificembodiment, the tumor may express elevated amounts of a growth factor,such as platelet derived growth factor, vascular endothelial growthfactor, or both. Also elevated levels of PDGF and VEGF could come fromthe tumor microenvironment due to angiogenic endothelial cells andvessels.

In yet another specific embodiment, the present invention provides amethod for treating a patient having a disease comprising excesscellular proliferation, excess angiogenesis, a tumor, or a combinationof any of the foregoing, wherein the method comprises administering aneffective amount of a pharmaceutical composition, wherein thepharmaceutical composition comprises at least one growth factor bindingcompounds or a pharmaceutically acceptable salt of any of the growthfactor binding compounds, and a pharmaceutically acceptable carrier; orone or more growth factor compounds, wherein the growth factor bindingcompounds comprise a plurality of acyclic isophthalic acid groupsattached to a non-peptide organic scaffold except for the compoundhaving the general structure:

wherein each R1 is:

and each R2 is:

Formulations (also referred to herein as compositions) include thosesuitable for local or systemic administration, such as oral, rectal,nasal, topical (including transdermal, buccal and sublingual), vaginal,parenteral (including subcutaneous, intramuscular, intravenous andintradermal) and pulmonary administration. The formulations canconveniently be presented in unit dosage form and can be prepared by anymethods well known in the art of pharmacy. Such methods include the stepof bringing into association the active ingredient with the carrierwhich constitutes one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers or both, and then if necessary shaping the product.Formulations of the present invention suitable for oral administrationcan be presented as discrete units such as capsules, cachets or tablets,each containing a predetermined amount of the active ingredient; or asan oil-in-water liquid emulsion, water-in-oil liquid emulsion or as asupplement within an aqueous solution, for example, a tea. The activeingredient can also be presented as bolus, electuary, or paste.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

Pharmaceutical compositions for topical administration according to thepresent invention can be formulated as an ointment, cream, suspension,lotion, powder, solution, paste, gel, spray, aerosol or oil.Alternatively, a formulation can comprise a patch or a dressing such asa bandage or adhesive plaster impregnated with active ingredients, andoptionally one or more excipients or diluents.

Formulations suitable for topical administration to the eye also includeeye drops wherein the active ingredient is dissolved or suspended in asuitable carrier, especially an aqueous solvent for the agent.

Formulations for rectal administration can be presented as a suppositorywith a suitable base comprising, for example, cocoa butter or asalicylate.

Formulation suitable for vaginal administration can be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the agent, such carriers as are known in theart to be appropriate.

Formulations suitable for nasal administration, wherein the carrier is asolid, include a coarse powder having a particle size, for example, inthe range of about 20 to about 500 microns, which is administered in themanner in which snuff is taken, i.e., by rapid inhalation through thenasal passage from a container of the powder held close up to the nose.Suitable formulations wherein the carrier is a liquid for administrationby nebulizer, include aqueous or oily solutions of the agent.

Formulations suitable for parenteral administration include aqueous andnon-aqueous isotonic sterile injection solutions that can containantioxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions that can include suspendingagents and thickening agents, and liposomes or other microparticulatesystems that are designed to target the compound to blood components orone or more organs. The formulations can be presented in unit-dose ormulti-does or multi-dose sealed containers, such as for example,ampoules and vials, and can be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carrier, forexample water for injections, immediately prior to use. Extemporaneousinjection solutions and suspensions can be prepared from sterilepowders, granules and tablets of the kind previously described.

Preferred unit dosage formulations are those containing a daily dose orunit, daily subdose, as herein above-recited, or an appropriate fractionthereof, of an agent. It should be understood that in addition to theingredients particularly mentioned above, the formulations of thisinvention can include other agents conventional in the art regarding thetype of formulation in question. For example, formulations suitable fororal administration can include such further agents as sweeteners,thickeners, and flavoring agents. It also is intended that the agents,compositions, and methods of this invention be combined with othersuitable compositions and therapies.

Various delivery systems are known and can be used to administer acompound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, receptor-mediated endocytosis and thelike. Methods of delivery include, but are not limited to,intra-arterial, intramuscular, intravenous, intranasal, and oral routes.In a specific embodiment, the pharmaceutical compositions of theinvention can be administered locally to the area in need of treatment;such local administration can be achieved, for example, by localinfusion during surgery, by injection, or by means of a catheter.

Therapeutic amounts can be empirically determined and will vary with thepathology being treated, the subject being treated, and the efficacy andtoxicity of the agent. Similarly, suitable dosage formulations andmethods of administering the agents can be readily determined by thoseof skill in the art.

The pharmaceutical compositions can be administered by any of a varietyof routes, such as orally, intranasally, parenterally or by inhalationtherapy, and can take form of tablets, lozenges, granules, capsules,pills, ampoule, suppositories or aerosol form. They can also take theform of suspensions, solutions, and emulsions of the active ingredientin aqueous or nonaqueous diluents, syrups, granulates or powders. Inaddition to a compound of the present invention, the pharmaceuticalcompositions can also contain other pharmaceutically active compounds ora plurality of compounds of the invention.

Ideally, the agent should be administered to achieve peak concentrationsof the active compound at sites of the disease. Peak concentrations atdisease sites can be achieved, for example, by intravenously injectingof the agent, optionally in saline, or orally administering, example, atablet, capsule or syrup containing the active ingredient.

Advantageously, the compositions can be administered simultaneously orsequentially with other drugs or biologically active agents, such asanti-cancer agents. Examples include, but are not limited to,antioxidants, free radical scavenging agents, peptides, growth factors,antibiotics, bacteriostatic agents, immunosuppressives, anticoagulants,buffering agents, anti-inflammatory agents, anti-pyretics, time-releasebinders, anesthetics, steroids and corticosteroids.

Preferably, the administering is carried out orally, parenterally,subcutaneously, intravenously, intramuscularly, intraperitoneally,intraarterially, transdermally or via a mucus membrane.

The term “cancer” is intended to mean any cellular malignancy whoseunique trait is the loss of normal controls which results in unregulatedgrowth, lack of differentiation and ability to invade local tissues andmetastasize. Cancer can develop in any tissue of any organ. Morespecifically, cancer is intended to include, without limitation,prostate cancer, leukemia, hormone dependent cancers, breast cancer,colon cancer, lung cancer, epidermal cancer, liver cancer, esophagealcancer, stomach cancer, cancer of the brain, and cancer of the kidney.

The terms “treatment”, “treating” and the like are intended to meanobtaining a desired pharmacologic and/or physiologic effect, e.g.,inhibition of cancer cell growth or induction of apoptosis of a cancercell. The effect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse effectattributable to the disease. “Treatment” as used herein covers anytreatment of a disease in a mammal, particularly a human, and includes:(a) preventing a disease or condition (e.g., preventing cancer) fromoccurring in an individual who may be predisposed to the disease but hasnot yet been diagnosed as having it; (b) inhibiting the disease, (e.g.,arresting its development); or (c) relieving the disease (e.g., reducingsymptoms associated with the disease).

The term “anti-cancer activity” is intended to mean an activity which isable to substantially inhibit, slow, interfere, suppress, prevent, delayand/or arrest a cancer and/or a metastasis thereof (such as initiation,growth, spread, and/or progression thereof of such cancer and/ormetastasis).

The terms “administering”, “administration”, and “contacting” areintended to mean a mode of delivery including, without limitation, oral,rectal, parenteral, subcutaneous, intravenous, intramuscular,intraperitoneal, intraarterial, transdermally or via a mucus membrane.The preferred one being orally. Administration may be carried outlocally, at a target site(s), or systemically. One skilled in the artrecognizes that suitable forms of oral formulation include, but are notlimited to, a tablet, a pill, a capsule, a lozenge, a powder, asustained release tablet, a liquid, a liquid suspension, a gel, a syrup,a slurry, a suspension, and the like. For example, a daily dosage can bedivided into one, two or more doses in a suitable form to beadministered at one, two or more times throughout a time period.

The term “therapeutically effective” is intended to mean an amount of acompound of the invention sufficient to substantially improve somesymptom associated with a disease or a medical condition. For example,in the treatment of cancer, a compound which decreases, prevents,delays, suppresses, or arrests any symptom of the disease would betherapeutically effective. A therapeutically effective amount of acompound is not required to cure a disease but will provide a treatmentfor a disease such that the onset of the disease is delayed, hindered,or prevented, or the disease symptoms are ameliorated, or the term ofthe disease is changed or, for example, is less severe or recovery isaccelerated in an individual.

The term “independently” is intended to mean that each of the four R1substituents and each of the four R2 substituents of the growth factorbinding compounds of the present invention may each be the samesubstituent or may each be a different substituent.

When the compounds of this invention are administered in combinationtherapies with other agents, they may be administered sequentially orconcurrently to an individual. Alternatively, pharmaceuticalcompositions according to the present invention may be comprised of acombination of a compound of the present invention, as described herein,and another therapeutic or prophylactic agent known in the art.

Pharmaceutically acceptable acid addition salts may be prepared frominorganic and organic acids. Salts derived from inorganic acids includehydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Salts derived from organic acids includecitric acid, lactic acid, tartaric acid, fatty acids, and the like.

Salts may also be formed with bases. Such salts include salts derivedfrom inorganic or organic bases, for example alkali metal salts such asmagnesium or calcium salts, and organic amine salts such as morpholine,piperidine, dimethylamine or diethylamine salts.

As used herein, the term “pharmaceutically acceptable carrier” includesany and all solvents (such as phosphate buffered saline buffers, water,saline), dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions. The pharmaceutical compositionsof the subject invention can be formulated according to known methodsfor preparing pharmaceutically useful compositions. Formulations aredescribed in a number of sources which are well known and readilyavailable to those skilled in the art. For example, Remington'sPharmaceutical Science (Martin E W (1995) Easton Pa., Mack PublishingCompany, 19^(th) ed.) describes formulations which can be used inconnection with the subject invention.

As used herein, the terms “individual” and “patient” are usedinterchangeably to refer to any vertebrate species, such as humans andanimals. Preferably, the patient is of a mammalian species. Mammalianspecies which benefit from the disclosed methods include, and are notlimited to, apes, chimpanzees, orangutans, humans, monkeys; domesticatedanimals (e.g., pets) such as dogs, cats, guinea pigs, hamsters,Vietnamese pot-bellied pigs, rabbits, and ferrets; domesticated farmanimals such as cows, buffalo, bison, horses, donkey, swine, sheep, andgoats; exotic animals typically found in zoos, such as bear, lions,tigers, panthers, elephants, hippopotamus, rhinoceros, giraffes,antelopes, sloth, gazelles, zebras, wildebeests, prairie dogs, koalabears, kangaroo, opossums, raccoons, pandas, hyena, seals, sea lions,elephant seals, otters, porpoises, dolphins, and whales. Human ornon-human animal patients can range in age from neonates to elderly.

In accordance with another embodiment of the present invention, there isprovided a method of treating cancer, comprising administering to anindividual a pharmaceutically effective amount of a pharmaceuticalcomposition of the present invention.

Preferably, a cancer to be treated in accordance with an embodiment ofthe present invention is selected from the group consisting of prostatecancer, leukemia, hormone dependent cancers, breast cancer, coloncancer, lung cancer, epidermal cancer, liver cancer, esophageal cancer,stomach cancer, cancer of the brain, and cancer of the kidney.

EXAMPLE 1 Identification of GFB204, a Calixarene Derivative thatPotently Inhibits VEGF and PDGF-Stimulation of Flk-1 and PDGF ReceptorTyrosine Phosphorylation

The initial approach to disrupt biologically significant protein-proteininteractions such as those involving growth factors with theirreceptors, consisted of designing molecules that contained foursynthetic peptide loops attached to a calix[4]arene scaffold (FIG. 1,reaction (a)) (Blaskovich, M. A. et al. Nat Biotechnol, 2000,18:1065-70). The peptide loop components were based on a cyclichexapeptide in which two residues are replaced by the dipeptide mimetic3-aminomethylbenzoate modified with a 5-amino group to provide linkageto the calixarene cavity. This design allowed the synthesis of a libraryof calixarene derivatives having different peptide sequences in theloops and large surface areas capable of binding protein surfaces. Oneof the library members, GFB 111, bound PDGF and blocked its binding toPDGFR at subμM concentrations and selectively relative to other growthfactors (Blaskovich, M. A. et al. Nat Biotechnol, 2000, 18:1065-70). Thefour peptide loops in GFB 111 contained negative and hydrophobicresidues in the sequence GDGY (FIG. 1, reaction (a)), which match wellwith the positive and hydrophobic amino acids in loops I, II and III ofthe homodimeric PDGF, which are critical for binding to PDGFR (Oefner,C. et al. Embo J, 1992, 11:3921-6; Andersson, M. et al. Growth Factors,1995, 12:159-64). GFB111 and similar compounds are described in U.S.Published Application No. U.S. 2003/0118589, filed Mar. 21, 2001, andInternational Published Application No. WO 01/70930, filed Mar. 21,2001, which are incorporated by reference in their entirety, includingall figures and tables. To improve this design, a second-generationlibrary has been designed in which in place of the peptide loops simple,acyclic isophthalic acid groups functionalized with a wide range ofacidic and hydrophobic groups (R₁ and R₂; FIG. 1, reaction (b); andTable 1) are attached to the calix[4]arene scaffold.

TABLE 1

IC₅₀ (uM) Compound R1 R2 PDGFR VEGFR GFB201

2.62 ± 0.5  4.96 ± 0.34 GFB202

1.39 ± 0.38 11.3 ± 2.90 GFB203

5.79 ± 0.72 19.83 ± 2.9  GFB204

0.19 ± 0.06 0.48 ± 0.31 GFB205

 3.1 ± 0.71 5.86 ± 0.68 GFB206

2.58 ± 0.21 >10>30 GFB207

3.47 ± 1.87 18.43 ± 2.72  GFB208

0.84 ± 0.06 >30>10 GFB209

2.91 ± 2.05 5.58 ± 4.11 GFB210

0.35 ± 0.31 4.54 ± 0.55 GFB211

1.34 ± 0.32 12.95 ± 0.49  GFB212

0.29 ± 0.08 >10>10>10 GFB213

0.57 ± 0.06 0.85 ± 0.44 GFB214

0.17 ± 0.02 >10>10>10 GFB215

0.21 ± 0.13 >10>10>10 GFB216

0.15 ± 0.01 >10>10>10 GFB217

>10>10 >10>10>10 GFB218

0.54 ± 0.15 >10>10>10 GFB219

0.53 ± 0.15 >10>10>10

To evaluate this library for molecules capable of preventing PDGF andVEGF binding to their receptors, their ability to disrupt PDGF- andVEGF-stimulated receptor tyrosine phosphorylation as described underMaterials and Methods was first determined. From the 19 compounds in thelibrary GFB204 was identified (where R₁ is a carboxylic acid and R₂ abenzyl ester) as a potent inhibitor of both VEGFR and PDGFR tyrosinephosphorylation (Table 1) with IC₅₀ values of 190 nM (PDGF) and 480 nM(VEGF). All the library members having at least four carboxylic groupsinhibited PDGF signaling at low μM concentrations (IC₅₀<6 μM), while theonly compound lacking acidic groups (GFB217) did not have a significantactivity. Analysis of the data in Table 1 revealed that all the potentinhibitors (having IC₅₀<0.6 μM) contain R₁=COOH and R₂=hydrophobic esteror amide. GFB211, where R₂=benzylamide, has an activity only slightlylower (IC₅₀=1.34±0.32 μM) than GFB204, while GFB209, (R₂=methyl ester)is less potent (IC₅₀=2.9±2.05 μM). These data suggest that the structureof the hydrophobic substituents is not crucial for PDGF signalinginhibition, as long as they are larger than a methyl group. Compoundshaving aromatic and aliphatic groups are equally active and the morestable amides have similar activity to their ester analogs.

In contrast, the calixarene derivatives containing amino acidsubstituents (i.e. GFB202, GFB203, GFB205, GFB206, GFB207 and GFB208)show in general lower activity (IC₅₀ in the range 1-6 μM). This may bedue either to a change in the ratio of ionic to hydrophobic groups onthe scaffold (these derivatives have 8-16 carboxylic acids and only 0-4hydrophobic substituents) or to a non-optimal distance between them.Moreover, the presence of acidic groups on the isophthalic spacer seemsto be more important than the presence of hydrophobic substituents:GFB201, GFB202, GFB203, and GFB206 lack hydrophobic groups in the R₁ andR₂ positions but are more potent than GFB217, which has no carboxylategroups. Possibly, the isophthalic acid groups themselves provide ahydrophobic area that interacts with the hydrophobic regions of thereceptors binding domain of PDGF.

Finally, it is important to note that the most active compounds in thestudy have exactly one carboxylic acid and one hydrophobic group on thefour isophthalic components within the scaffold consistent with previousstudies which led to the identification of GFB111 (Blaskovich, M. A. etal. Nat Biotechnol, 2000, 18:1065-70). SAR studies also revealed thatthe characteristics necessary in this series for inhibition ofVEGF-stimulated Flk-1 tyrosine phosphorylation are much more stringent.Indeed, besides GFB204 (IC₅₀=0.48±0.31 μM), only one other potentcompound, GFB213, inhibited Flk-1 tyrosine phosphorylation with an IC₅₀value of 0.85±0.44 μM (Table 1). The factors that determine theinhibition activity towards VEGF signaling are difficult to infer fromthe data in Table 1.

GFB204 binds both PDGF and VEGF. The ability of GFB204 to bind both VEGFand PDGF was demonstrated by fluorescence titration curves. Both VEGFand PDGF contain tryptophans that fluoresce at 334 nM when excited at294 nM. FIGS. 2D and 2E show that increasing concentrations of GFB-204decreased the ability of PDGF and VEGF to fluoresce in a concentrationdependent manner.

EXAMPLE 2 GFB204 Inhibits VEGF and PDGF but not EGF Binding to theirRespective Receptors

The ability of GFB204 to inhibit PDGF and VEGF-stimulated receptortyrosine phosphorylation suggested that GFB204 either disruptsligand/receptor binding, receptor dimerization or receptor tyrosinekinase activity. Therefore, it was determined whether GFB204 inhibitsthe interaction between PDGF and VEGF and their respective receptors butnot other growth factors. To this end, the present inventors evaluatedthe ability of GFB204 to block [I-125]-PDGF, [I-125]-VEGF and [I-125]EGFbinding to their receptor on NIH 3T3 cells (PDGF), NIH 3T3 cellsoverexpressing human Flk-1 (VEGF) and human EGFR (EGF) as describedunder Materials and Methods. GFB204 effectively inhibited the binding of[I-125]PDGF and [I-125]-VEGF to their receptors with IC₅₀ values of154+/−1.0 nM and 469 +/−94 nM, respectively (FIGS. 2A-2C). In contrast,[I-125]EGF binding to its receptor was not affected by GFB204 withconcentrations as high as 100 μM. Thus, GFB204 is more selective forPDGF and VEGF over EGF.

EXAMPLE 3 GFB204 Disrupts PDGF- and VEGF- but not EGF-, bFGF- or IGF-1Stimulation of Erk1, Erk2, Akt and STAT3 Phosphorylation

To further document the selectivity of GFB204 for PDGF and VEGF overother growth factors, the present inventors determined the ability ofGFB204 to block growth factor stimulation of the kinases Erk1, Erk2 andAkt as well as the signal transducer and activator of transcriptionSTAT3. To this end, NIH 3T3 cells (PDGF and bFGF) or NIH 3T3 cells thatoverexpress Flk-1 (VEGF), EGFR (EGF) or IGF-R (IGF-1) were starved andstimulated with the corresponding growth factor in the presence orabsence of GFB204, and the cells were processed for anti-phosphotyrosine(PDGF and VEGF) and for anti-phospho-Erk1/2, Akt and STAT3 (PDGF, VEGF,EGF, bFGF and IGF-1) Western immunoblotting as described under Materialsand Methods. FIG. 3A shows that, as described in Table 1, treatment ofstarved cells with PDGF or VEGF resulted in potent stimulation ofreceptor tyrosine phosphorylation and that treatment with GFB204inhibited this stimulation with IC₅₀ values of 190 nM and 480 nM,respectively. Similarly, PDGF- and VEGF-stimulation of Erk1 Erk2 wasalso inhibited with similar IC₅₀ values. Furthermore, this inhibitionwas selective in that GFB204 blocked PDGF- and VEGF- but had littleeffect on EGF-, bFGF- and IGF-1-stimulation of the phosphorylation ofErk1, Erk2, Akt and STAT3 (FIG. 3B).

EXAMPLE 4 GFB204 Inhibits Angiogenesis In Vitro and In Vivo andSuppresses the Growth of Human Tumors in Nude Mice

The ability of GFB204 to inhibit potently and selectively PDGF andVEGF/ligand receptor binding and subsequent signaling prompted thepresent inventors to determine whether this agent could inhibitangiogenesis, in vitro and in vivo and subsequently inhibit tumorgrowth. First, it was determined if GFB204 could inhibit angiogenesis invitro by evaluating its ability to suppress VEGF-induced human brainendothelial capillary network formation as described under Materials andMethods. GFB204 was highly efficient at inhibiting VEGF-inducedcapillary network formation with an IC₅₀ value of 700 nM (FIG. 4C). Theability of GFB204 to inhibit human brain endothelial cell migration asdescribed under Materials and Methods was determined next. GFB204inhibited VEGF-induced endothelial cell migration through matrigel poresinto the lower chamber with an IC₅₀ value of 600 nM (FIG. 4B).

The ability of GFB204 to inhibit VEGF and PDGF binding to theirreceptors and subsequent signaling coupled with its ability to inhibitVEGF-induced endothelial cell migration and capillary network formationsuggested that GFB204 might inhibit angiogenesis and tumorigenesis inwhole animals. Therefore, it was next evaluated whether GFB204 is ableto suppress tumor growth and angiogenesis in vivo by implanting humanlung cancer A-549 cells s.c. in nude mice. When tumors reached anaverage size of 100 mm³, the mice were treated with either vehicle orGFB204 and 3 weeks later the tumors were removed and processed for CD31immunostaining to determine GFB204 anti-angiogenic effects as describedunder Materials and Methods. Tumors from control animals grew to anaverage size of 749±111 mm³ (FIG. 6). In contrast, tumors fromGFB204-treated animals grew to an average size of only 650±114 mm³(GFB204; 1 mg/kg), and 284±108 mm³ (GFB204; 5 mg/kg), respectively.Thus, treatment with GFB204 resulted in a statistically significant(p<0.05), tumor growth inhibition at 5 mg/kg (73%), but not at 1 mg/kg(15%). Tumor sections from GFB204 treated animals show a significantinhibition of CD31 staining (FIGS. 5A-5C). Quantification ofmicrovessels at field magnification (400×) indicated that tumors fromvehicle-treated mice contained 11.3±1.9 microvessels whereas those frommice treated with GFB204 (5 mg/Kg) had only 2.6±0.9 microvessels. Takentogether, the results clearly demonstrated that GFB204 inhibits A-549xenografts tumor growth and angiogenesis in vivo.

The strict requirement and stringent dependence of tumor growth onangiogenesis has prompted many investigators to design strategies forcancer therapy by disrupting angiogenesis resulting in deprivation ofcancer cells of nutrients and essentially tumor starvation (Zhang, W. etal. Angiogenesis, 2002, 5:35-44; Ferrara, N. Semin Oncol, 2002, 29:10-4;Jain, R. K. Semin Oncol, 2002, 29:3-9; Morin, M. J. Oncogene, 2000,19:6574-83; Miao, R. Q. et al. Blood, 2002, 100:3245-52; Laird, A. D. etal. Cancer Res, 2000, 60:4152-60; Wedge, S. R. et al. Cancer Res, 2000,60:970-5; Relf, M. et al. Cancer Res, 1997, 57:963-9; Huang, J. et al.Proc Natl Acad Sci USA, 2003, 100:7785-90; Blaskovich, M. A. et al. NatBiotechnol, 2000, 18:1065-70). Although targeting angiogenesis as anapproach to cancer therapy was suggested decades ago, it is only veryrecently that the first drug designed to target a step in the complexprocess of angiogenesis has been approved by the FDA (Ferrara, N. SeminOncol, 2002, 29:10-4). Indeed, AVASTIN, a humanized anti-VEGF monoclonalantibody has shown activity against metastatic colon cancer. Thoughpivotal for providing proof of concept for targeting angiogenesis inhumans, this approach has not been fully exploited. One improvement thatis sought after is to design strategies that simultaneously targetdifferent steps in the angiogenic process (Bergers, G. et al. J ClinInvest, 2003, 111: 1287-95; Relf, M. et al. Cancer Res, 1997, 57:963-9;Huang, J. et al. Proc Natl Acad Sci USA, 2003, 100:7785-90). The presentinventors have developed a novel synthetic pharmacological agent thatinhibits the function of both VEGF and PDGF, growth factors that havebeen shown to mediate initiation and maintenance of new blood vessels,respectively (Bergers, G. et al. J Clin Invest, 2003, 111:1287-95;Dvorak, H. F. J Clin Oncol, 2002, 20:4368-80; Ferrara, N. Curr TopMicrobiol Immunol, 1999, 237:1-30; Dvorak, H. F. et al. Curr TopMicrobiol Immunol, 1999, 237:97-132; Eriksson, U. and Alitalo, K. CurrTop Microbiol Immunol, 1999, 237:41-57). This is the first report of anagent that inhibits the binding of both VEGF and PDGF to their receptorsand subsequently suppresses tyrosine phosphorylation and downstreamsignaling pathways (Erk, Akt and STAT3). GFB204 also blocked potentlythe ability of endothelial cells to migrate (IC₅₀=600 nM) as well astheir ability to form capillaries in vitro (IC₅₀=700 nM). In vivo,treatment of mice bearing human tumors s.c. led to inhibition of bloodvessel formation around the tumor mass as well as inhibition of tumorgrowth. Although GFB204 potently inhibited both VEGF and PDGF binding totheir receptors (200-500 nM) it was not a non-specific disrupter of allof ligand/receptor binding since EGF binding to its receptor was notaffected at doses as high as 100 μM. Further support for selectivity wasprovided by demonstrating that GFB204 inhibited the activation of Erk1,Erk2, Akt and STAT3 by PDGF and VEGF but not by EGF, bFGF or IGF-1.

Identification of calix[4]arene derivatives capable of blocking bindingof both VEGF and PDGF to their receptors is an entirely novel approachto targeting receptor tyrosine kinase signaling. Although the anti-VEGFantibody AVASTIN also blocks VEGF binding to its receptor (Zhang, W. etal. Angiogenesis, 2002, 5:35-44; Ferrara, N. Semin Oncol, 2002,29:10-4), there are apparently no other agents that block binding ofboth PDGF and VEGF to their receptors. Furthermore, the advantage ofGFB204 over AVASTIN is that GFB204 is a much smaller molecule that canbe easily synthesized at low cost, unlike the laborious and expensivemethods involved in generating antibodies for therapeutic purposes.Although prior to this report there were no dual inhibitors of VEGF andPDGF binding to their receptors, dual inhibitors of VEGF and PDGFreceptor tyrosine kinases have been made and some are in clinical trials(Kerbel, R. S. Carcinogenesis, 2000, 21:505-15; Jain, R. K. Semin Oncol,2002, 29:3-9; Morin, M. J. Oncogene, 2000, 19:6574-83; Miao, R. Q. etal. Blood, 2002, 100:3245-52; Laird, A. D. et al. Cancer Res, 2000,60:4152-60; Wedge, S. R. et al. Cancer Res, 2000, 60:970-5).

There are distinct differences between these ATP mimics and GFB204.While the target for GFB204 is the ligand/receptor interaction thatoccurs extracellularly on the outer cell surface, ATP mimics target thetyrosine kinase domains of the receptors that are intracellular.Therefore, unlike GFB204, kinase inhibitors must enter cells to reachtheir target. Furthermore, most tyrosine kinase inhibitors target theATP binding site, variations of which are ubiquitous in cells.Therefore, the outcome of treating patients with GFB204 may be verydifferent from that of treating patients with an ATP mimic that targetsboth PDGF and VEGF receptor tyrosine kinases. Advanced preclinicalstudies are underway in preparation for an IND application for phase Itesting of GFB204 in humans.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication.

1. A growth factor binding compound according to structure (I):

wherein each R1 is independently selected from the group consisting of:

wherein each R2 is independently selected from the group consisting of:


2. The growth factor binding compound according to claim 1, wherein thecompound is selected from the group consisting of compounds according tostructure (I), wherein: each R1 is

and each R2 is

each R1 is

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3. The growth factor binding compounds according to claim 1, wherein thecompound is capable of binding to platelet derived growth factors,vascular endothelial growth factor, or a mixture of any of theforegoing.
 4. A pharmaceutical composition comprising one or more growthfactor binding compounds of claim 1 or a pharmaceutically acceptablesalt of any of the growth factor binding compounds, and apharmaceutically acceptable carrier.
 5. The pharmaceutical compositionaccording to claim 4, wherein each of the growth factor bindingcompounds are selected from the group consisting of compounds accordingto structure (I), wherein: each R1 is

and each R2 is

each R1 is

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6. The pharmaceutical composition according to claim 4, wherein each ofthe growth factor binding compounds are capable of binding to plateletderived growth factors, vascular endothelial growth factor, or a mixtureof any of the foregoing.
 7. The growth factor compound of claim 1,wherein each R1 is:

and each R2 is:


8. The pharmaceutical composition according to claim 4, wherein each R1is:

and each R2 is: